DNA molecules encoding fibrinolytically active polypeptide

ABSTRACT

A fibrinolytically active metalloproteinase polypeptide (called “novel acting thrombolytic”) which is useful for blood clot lysis in vivo and methods and materials for its production by recombinant expression are described.

This application is a divisional of U.S. Ser. No. 09/411,329, filed Oct. 1, 1999 now issued as U.S. Pat. No. 6,261,820, which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a fibrinolytically active metalloproteinase of non-naturally occurring sequence, to combinant methods for its manufacture, and to its use in treating thrombosis in vivo.

BACKGROUND OF THE INVENTION

Fibrolase is an enzymatically active polypeptide (specifically, a metalloproteinase) composed of 203 amino acid residues that was originally isolated by purification from the venom of the Southern Copperhead snake; U.S. Pat. No. 4,610,879, issued Sep. 9, 1986 (Markland et al.); and Guan et al., Archives of Biochemistry and Biophysics, Volume 289, Number 2, pages 197-207 (1991). The enzyme exhibits direct fibrinolytic activity with little or no hemorrhagic activity, and it dissolves blood clots made either from fibrinogen or from whole blood.

The amino acid sequence of fibrolase has also been determined, with methods described for recombinant production in yeast and use for the treatment of thrombembolic conditions in vivo; Randolph et al., Protein Science, Cambridge University Press (1992), pages 590-600, and European Patent Application No. 0 323 722 (Valenzuela et al.), published Jul. 12, 1989.

SUMMARY OF THE INVENTION

This invention provides a fibinolytic metalloproteinase having the non-naturally occurring linear array of amino acids depicted in SEQ ID NO: 1, also referred to herein as “novel acting thrombolytic” (or “NAT”). Also provided are nucleic acid molecules, such as the one of SEQ ID NO: 2 and variants thereof encoding NAT.

The term “mature” is used in its conventional sense to refer to the biologically active polypeptide which has been enzymatically processed in situ in the host cell to cleave it from the prepro region.

Because of its fibrinolytic activity, NAT is useful in vivo as a blood clot lysing agent to treat thrombosis in a mammal (including rats, pigs and humans).

The NAT polypeptide of this invention provides advantages over naturally occurring fibrolase as a therapeutic agent (i.e., the fibrinolytic polypeptide found in snake venom). Native fibrolase is known to contain several alternate N-termini: QQRFP (amino acids 1-5 of SEQ ID NO:5), EQRFP (amino acids 1-5 of SEQ ID NO:15) and ERFP (amino acids 1-4 of SEQ ID NO:16) (in which “E” designates a cyclized glutamine, or pyroglutainic acid). More specifically, starting with an N-terminus composed of QQRFP (see SEQ ID NO:5), the fibrolase molecule undergoes degradation to result in two isoforms, having N-terminal sequences of EQRFP (see SEQ ID NO:15) and ERFP (see SEQ ID NO:16), respectively. Recombinant fibrolase as produced in yeast typically yields a mixture of all three of these forms and is thus not homogeneous. See Loayza et al., Journal of Chromatography, B 662, pages 227-243 (1994). Moreover, the cyclized glutamine residue results in a “blocked” N-terminus which makes sequencing impossible.

In contrast, the recombinant NAT of this invention provides a single species: only one N-terminus is typically produced. The result is greater homogeneity of the end product compared to recombinant fibrolase, which is beneficial when medical applications are the intended end use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts in linear fashion the full amino acid sequence of NAT (SEQ ID NO: 3), consisting of the “prepro” region (underscored), which includes the “signal” peptide, and the mature polypeptide (non-underscored).

DETAILED DESCRIPTION OF THE INVENTION

NAT may be produced by recombinant expression of the nucleic acid molecule of SEQ ID NO: 4, which encodes the full amino acid sequence of NAT (SEQ ID NO: 3), including the prepro region from nucleotides 1-783 and mature polypeptide from nucleotides 784-1386, in a suitable host. Following expression, the prepro region is enzymatically processed off in the host cell to yield the mature active polypeptide (SEQ ID NO: 1).

Preferably, NAT is produced recombinantly in yeast, as will be explained in greater detail further below.

The mature polypeptide (SEQ ID NO: 1) which is thus produced may or may not have an amino terminal methionine, depending on the manner in which it is prepared. Typically, an amino terminal methionine residue will be present when the polypeptide is produced recombinantly in a non-secreting bacterial (e.g., E. coli) strain as the host.

Besides the nucleic acid molecule of SEQ ID NO: 2, also utilizable are degenerate sequences thereof which encode the same polypeptide. The present invention also embraces nucleic acid molecules that may encode additional amino acid residues flanking the 5′ or 3′ portions of the region encoding the mature polypeptide, such as sequences encoding alternative pre/pro regions (i.e., sequences responsible for secretion of the polypeptide through cell membranes) in place of the “native” pre/pro region (i.e., found in naturally occurring fibrolase). The additional sequences may also be noncoding sequences, including regulatory sequences such as promoters of transcription or translation, depending on the host cell.

NAT can be prepared using well known recombinant DNA technology methods, such as those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and/or Ausubel et al., editors, Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, New York (1994). A DNA molecule encoding the polypeptide or truncated version thereof may be obtained, for example, by screening a genomic or cDNA library, or by PCR amplification, to obtain a nucleic acid molecule encoding fibrolase, followed by replacement of the codons encoding the N-terminal amino acid residues QQR with a codon for serine (S). Alternatively, a DNA molecule encoding NAT may be prepared by chemical synthesis using methods well known to the skilled artisan, such as those described by Engels et al. in Angew. Chem. Intl. Ed., Volume 28, pages 716-734 (1989). Typically, the DNA will be several hundred nucleotides in length. Nucleic acids larger than about one hundred nucleotides can be synthesized as several fragments using these same methods and the fragments can then be ligated together to form a nucleotide sequence of the desired length.

The DNA molecule is inserted into an appropriate expression vector for expression in a suitable host cell. The vector is selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, such that expression of the DNA can occur). The polypeptide may be expressed in prokaryotic, yeast, insect (baculovirus systems) or eukaryotic host cells, although yeast is preferred as will be explained in greater detail further below.

The vectors used in any of the host cells to express NAT may also contain a 5′ flanking sequence (also referred to as a “promoter”) and other expression regulatory elements operatively linked to the DNA to be expressed, as well as enhancer(s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding NAT, and a selectable marker element. Each of these elements is discussed below.

Optionally, the vector may also contain a “tag” sequence, i.e., an oligonucleotide sequence located at the 5′ or 3′ end of the polypeptide-coding sequence that encodes polyHis (such as hexaHis) or another small immunogenic sequence (such as the c-myc or hemagglutinin epitope, for which antibodies, including monoclonal antibodies, are commercially available). This tag will be expressed along with NAT, and can serve as an affinity tag for purification of this polypeptide from the host cell. Optionally, the tag can subsequently be removed from the purified polypeptide by various means, for example, with use of a selective peptidase.

The 5′ flanking sequence may be the native 5′ flanking sequence, or it may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of 5′ flanking sequences from more than one source), or synthetic. The source of the 5′ flanking sequence may be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the 5′ flanking sequence is functional in, and can be activated by the host cell machinery.

The origin of replication element is typically a part of prokaryotic expression vectors purchased commercially and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for optimal expression of NAT. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and then ligated into the vector.

The transcription termination element is typically located 3′ to the end of the polypeptide coding sequence and serves to terminate transcription of the mRNA. Usually, the transcription termination element in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While the element is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using known methods for nucleic acid synthesis.

A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that: (a) confer resistance to antibiotics or other toxins, for example, ampicillin, tetracycline or kanamycin for prokaryotic host cells, zeocin for yeast host cells, and neomycin for mammalian host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. Preferred selectable markers for use in prokaryotic expression are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarno sequence (for prokaryotes) or the Kozak sequence (for eukaryotes), is necessary for the initiation of translation of mRNA. The element is typically located 3′ to the promoter and 5′ to the coding sequence of the polypeptide to be synthesized. The Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector. The Kozak sequence typically includes sequences immediately before and after the initiating codon. A preferred Kozak sequence is one that is associated with a high efficiency of initiation of translation at the AUG start codon.

In those cases where it is desirable for NAT polypeptide to be secreted from the host cell, a signal sequence may be used to direct the polypeptide out of the host cell where it is synthesized. Typically, the signal sequence is positioned in the coding region of nucleic acid sequence, or directly at the 5′ end of the coding region. Many signal sequences have been identified, and any of them that are functional in the selected host cell may be used here. Consequently, the signal sequence may be homologous or heterologous to the polypeptide. Additionally, the signal sequence may be chemically synthesized using methods referred to above.

After the vector has been constructed and a nucleic acid has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.

As mentioned, host cells may be prokaryotic (such as E. coli) or eukaryotic (such as a yeast cell, an insect cell, or a vertebrate cell). The host cell, whether it be yeast or some other host, when cultured under appropriate conditions can synthesize NAT, which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). After collection, NAT polypeptide can be purified using methods such as molecular sieve chromatography, affinity chromatography, and the like.

Selection of the host cell will depend in large part on whether the manner in which the host cell is able to “fold” NAT into its native secondary and tertiary structure (e.g., proper orientation of disulfide bridges, etc.) such that biologically active material is prepared by the cell. However, even where the host cell does not synthesize biologically active material, it may be “folded” after synthesis using appropriate chemical conditions, such as ones that are known to those skilled in the art. In either case, proper folding can be inferred from the fact that biologically active material has been obtained.

Suitable host cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. Other suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene. Still other suitable mammalian cell lines include but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

Also useful as host cells are bacterial cells. For example, the various strains of E. coli (e.g., HB101, DH5a, DH10, and MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and the like, may also be employed. Additionally, many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptide of the present invention. Also, where desired, insect cells may be utilized as host cells. See, for example, Miller et al., Genetic Engineering, Volume 8, pages 277-298 (1986).

Insertion (also referred to as “transformation” or “transfection”) of the vector into the selected host cell may be accomplished using such methods as calcium phosphate, electroporation, microinjection, lipofection or the DEAE-dextran method. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al., above.

The host cells containing the vector may be cultured using standard media well known to the skilled artisan. The media will usually contain all nutrients necessary for the growth and survival of the cells. Suitable media for culturing E. coli cells are, for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular cell line being cultured. A suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate and/or fetal calf serum, as necessary.

Typically, an antibiotic or other compound useful for selective growth of the transformed cells only is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin.

The amount of NAT produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.

If NAT is secreted from the host cells other than gram-negative bacteria, the majority will likely be found in the cell culture medium. If NAT is secreted from gram-negative bacteria, it will to some degree be found in the periplasm. If NAT is not secreted, it will be present in the cytoplasm.

For intracellular NAT, the host cells are typically first disrupted mechanically. For NAT having a periplasmic location, either mechanical disruption or osmotic treatment can be used to release the periplasmic contents into a buffered solution. NAT polypeptide is then isolated from this solution. Purification from solution can thereafter be accomplished using a variety of techniques. If NAT has been synthesized so that it contains a tag such as hexahistidine or other small peptide at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i.e., a monoclonal antibody). For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen nickel columns) can be used for purification. (See, for example, Ausubel et al., editors, Current Protocols in Molecular Biology, above).

Where, on the other hand, the polypeptide has no tag and no antibodies are available, other well known procedures for purification can be used. Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, reversed phase chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing (ISOPRIME® iso-electric protein purification apparatus, Hoefer Pharmacia Biotech Inc.). In some cases, two or more of these techniques may be combined to achieve increased purity.

Especially preferred for use in the production of NAT are yeast cells, and most advantageously those of the yeast genus known as Pichia (e.g., Pichia pastoris), because of the greater efficiency of refolding compared to, for instance, bacterial cells such as E. coli. Suitable recombinant methods of expression for this yeast strain are described in U.S. Pat. Nos. 4,855,231 (Stroman et al.), 4,812,405 (Lair et al.), 4,818,700 (Cregg et al.), 4,885,242 (Cregg) and 4,837,148 (Cregg), the disclosures of which are incorporated herein by reference.

Notably, Pichia cells can also be used to express fibrolase with similar efficiency from DNA molecules encoding this metalloproteinase, and such a method constitutes an additional aspect of the present invention. Fibrolase is a known metalloproteinase which has been described in the scientific and patent literature; see Randolph et al., and European Patent Application No. 0 323 722, cited above. Typically, the fibrolase to be expressed will be of SEQ ID NO: 5, which is encoded by the cDNA molecule of SEQ ID NO: 6 (or variants thereof encoding the same amino acid sequence). The expression of fibrolase in such a system will typically involve a DNA molecule of SEQ ID NO: 7, which encodes “prepro” sequence (nucleotides 1-783) in addition to the “mature” polypeptide (nucleotides 784-1392).

Chemically modified versions of NAT in which the polypeptide is linked to a polymer or other molecule to form a derivative in order to modify properties are also included within the scope of the present invention. For human therapeutic purposes especially, it may be advantageous to derivatize NAT in such a manner by the attachment of one or more other chemical moieties to the polypeptide moiety. Such chemical moieties may be selected from among various water soluble polymers. The polymer should be water soluble so that the NAT polypeptide to which it is attached is miscible in an aqueous environment, such as a physiological environment. The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random or non-random copolymers (see further below regarding fusion molecules), and dextran or poly(n-vinyl pyrolidone)polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, polystyrenemaleate and polyvinyl alcohol. Polyethylene glycol propionaldenhyde may have advantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 2 kilodaltons (kDa) and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol on a therapeutic protein).

The number of polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols). The proportion of polymer molecules to NAT polypeptide molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted polypeptide or polymer) will be determined by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.

The chemical moieties should be attached to NAT with consideration of effects on functional or antigenic domains of the polypeptide. There are a number of attachment methods available to those skilled in the art. See, for example, EP 0 401 384 (coupling PEG to G-CSF), and Malik et al., Experimental Hematology, Volume 20, pages 1028-1035 (1992) (reporting the pegylation of GM-CSF using tresyl chloride). By way of illustration, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule (or other chemical moiety) may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residue. Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s) (or other chemical moiety). Preferred for manufacturing purposes is attachment at an amino group, such as at the N-terminus or to a lysine group. Attachment at residues important for receptor binding should be avoided if receptor binding is desired.

One may specifically desire N-terminally chemically modified derivatives. Using polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to polypeptide molecules in the reaction mixture, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated NAT. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated NAT molecules. Selective N-terminal chemical modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization. See PCT application WO 96/11953, published Apr. 25, 1996. Under the appropriate reaction conditions, substantially selective derivatization of NAT at the N-terminus with a carbonyl group containing polymer is achieved. For example, one may selectively N-terminally pegylate NAT by performing the reaction at a pH which allows one to take advantage of the pK_(a) differences between the ε-amino group of the lysine residues and that of the α-amino group of the N-terminal residue of the polypeptide. By such selective derivatization, attachment of a polymer to a polypeptide is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the polypeptide and no significant modification of other reactive groups, such as lysine side chain amino groups, occurs. Using reductive alkylation, the polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the polypeptide. Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.

NAT or chemically modified derivatives in accordance with the invention may be formulated for in vivo administration, and most preferably via intra-thrombus (i.e., via localized delivery directly to the site of the clot in the blood vessel, e.g., as by catheter). Systemic delivery is normally not preferred due to the likelihood that innate α2 macroglobulin in the general circulation may complex with NAT to prevent interaction with fibrin or fibrinogen, thus impairing clot lysis. However, there may be instances where larger amounts of NAT can be used which exceed the circulating levels of α2 macroglobulin, thus enabling systemic administration and delivery. In general, encompassed within the invention are pharmaceutical compositions comprising effective amounts of NAT together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. By “effective amount” is meant amount sufficient to produce a measurable biological effect (i.e., a thrombolytically effective amount which effects lysis of the blood clot or clots being treated).

Typically, NAT will be in highly purified form, and any pharmaceutical composition being used as the delivery vehicle will normally be presterilized for use, such as by filtration through sterile filtration membranes.

One skilled in the art will be able to ascertain effective dosages by administration and observing the desired therapeutic effect. Particular effective doses within this range will depend on the particular disorder or condition being treated, as well as the age and general health of the recipient, and can be determined by standard clinical procedures. Where possible, it will be desirable to determine the dose-response curve of the pharmaceutical composition first in vitro, as in bioassay systems, and then in useful animal model systems in vivo prior to testing in humans. The skilled practitioner, considering the therapeutic context, type of disorder under treatment, and other applicable factors, will be able to ascertain proper dosing without undue effort. Typically, a practitioner will administer the NAT composition until a dosage is reached that achieves the desired effect (i.e., lysis of the blood clot). The composition may be administered as a single dose, or as two or more doses (which may or may not contain the same amount of polypeptide) over time, or on a continuous basis.

NAT may also be used to generate antibodies in accordance with standard methods. The antibodies may be polyclonal, monoclonal, recombinant, chimeric, single-chain and/or bispecific, etc. To improve the likelihood of producing an immune response, the amino acid sequence of NAT can be analyzed to identify portions of the molecule that may be associated with increased immunogenicity. For example, the amino acid sequence may be subjected to computer analysis to identify surface epitopes, such as in accordance with the method of Hope and Woods, Proceedings of the National Academy of Science USA, Volume 78, pages 3824-3828 (1981).

Various procedures known in the art can be used for the production of polyclonal antibodies which recognize epitopes of NAT. For the production of antibody, various host animals can be immunized by injection with the polypeptide, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's, mineral gels such as aluminum hydroxide (alum), surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum.

For the preparation of monoclonal antibodies directed toward NAT, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein which is described in Nature, Volume 256, pages 495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique described by Kozbor et al. in Immunology Today, Volume 4, page 72 (1983), and the EBV-hybridoma technique to produce monoclonal antibodies described by Cole et al. in “Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss, Inc., pages 77-96 (1985), are all useful for preparation of monoclonal antibodies in accordance with this invention.

The antibodies of this invention can be used therapeutically, such as to bind to and thereby neutralize or inhibit excess amounts of NAT in vivo after administration. The antibodies can further be used for diagnostic purposes, such as in labeled form to detect the presence of NAT in a body fluid, tissue sample or other extract, in accordance with known diagnostic methods.

Description of Specific Embodiments

The invention is further illustrated in the following examples.

EXAMPLE 1 Derivation of NAT Sequence

An effective way to produce fibrolase is to express it initially as preprofibolase in which cleavage by the protease kex-2 occurs at the junction of the “prepro” and “mature” regions to yield biologically active material (“mature” fibrolase). From this design, the synthesis and processing of the preprofibrolase leads to secretion of mature fibrolase into the culture medium. The actual sequence at the cleaved junction is ( . . . TKR≅QQRF . . . ) comprising amino acids 259-265 of SEQ ID NO:14.

Kex-2 is an endoprotease that cleaves after two adjacent basic amino acids, in this case lysine (K)-arginine(R). Mature fibrolase expressed from DNA having the above mentioned sequence revealed that the expected N-terminal glutamine (Q) residue had in fact undergone deamidation and cyclization to generate pyroglutamic acid (E). This chemical modification was deemed undesirable, since peptides with an N-terminal cyclized glutamine (pyroglutamic acid) residue fail to react in the Edman degradation procedure for amino acid sequencing. Accordingly, both of the glutamine (Q) residues at the N-terminus in the sequence for mature fibrolase (amino acids 1-2 OF SEQ ID NO:5) were deleted, resulting in an N-terminal arginine (R) residue. Since kex-2 cleaves after two adjacent basic amino acids as mentioned, it was anticipated that the sequence ( . . . KRRF . . . ) (SEQ ID NO:21) would present an ambiguous site for kex-2 cleavage. Accordingly, the N-terminal arginine (R) residue (shown underlined above) was replaced with a serine (S) residue to result in the sequence ( . . . KRSF . . . ); see amino acids 260-263 of SEQ ID NO:17. The choice of serine was based on the need to introduce an amino acid which facilitates kex-2 cleavage when it occurs on the C-terminal side of the hydolysis site. Rholam et al., European Journal of Biochemistry, Volume 227, pages 707-714 (1995)

As a result, the DNA sequence for preprofibrolase was modified by site-directed mutagenesis at the N-terminal coding region for mature fibrolase to substitute the codons for “QQR” with a codon for “S”, using a standard PCR protocol, thus resulting in preproNAT having the amino acid sequence of SEQ ID NO: 3. The oligonucleotides used to prime the PCR reactions are listed below, and their homology with the target sequence is also shown. Initially, two PCR reactions were carried out using oligos 1 and 4 as one primer pair and oligos 2 and 3 as another primer pair, both with DNA of the parent gene as the template. The DNA products of these two reactions (602 and 816 nucleotides in length, which are SEQ ID NOs:18 and 19, respectively,) were purified by agarose gel electrophoresis and combined to serve as a template in a second round of PCR using oligos 1 and 2 as the primer pair. This final PCR product (1373 nucleotides in length, which is SEQ ID NO:20,) was cleaved with restriction endonucleases XhoI and NotI. The digest was deproteinized with phenol/chloroform and DNA precipitated. A portion of the recovered DNA was ligated into the plasmid pPICZα (Invitrogen, Carlsbad, Calif., Catalog No. VI95-20), which had been similarly cleaved with restriction endonucleases XhoI and NotI, enzymatically dephosphorylated, and deproteinized with phenol/chloroform. All subsequent steps were carried out according to the Invitrogen Pichia Expression Kit manual (Invitrogen Corp., Catalog No. K1710-01). The ligation reaction products were transformed into E. coli by electroporation and selected for survival on zeocin-containing solid media. The plasmid was isolated and the profibrolase region was confirmed by DNA sequencing. The plasmid was linearized by cleaving with restriction endonuclease PmeI and then transformed into Pichia pastoris GS115his⁺. The GS115 strain is normally his, so the his genotype was restored by transformation with a DNA source carrying the wild type version of the his4 gene. Alternatively, a his⁺ strain can be obtained commercially from Invitrogen Corp. (X-33 cell line, Catalog No. C180-00). Integrants were selected as zeocin-resistant colonies. Candidate clones were induced in methanol-containing media, and the broth was assayed for NAT production on 4-20% PAGE, using Coomassie staining.

Oligo 1 5′-TACTATTGCCAGCATTGCTGC-3′ (SEQ ID NO: 8) Oligo 2 5′-GCAAATGGCATTCTGACATCC-3′ (SEQ ID NO: 9) Oligo 3 (SEQ ID NO: 10) 5′-TCCAATTAAACTTGACTAAGAGATCTTTCCCACAAAGATACGTAC-3′ Oligo 4 (SEQ ID NO: 11) 5′-GTACGTATCTTTGTGGGAAAGATCTCTTAGTCAAGTTTAATTGG-3′

The location of these oligonucleotides is shown below in relation to the double-stranded DNA sequence (SEQ ID NO: 12 coding or sense strand, SEQ ID NO: 13 complementary or antisense strand) and corresponding amino acid sequences (SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO. 23, SEQ ID NO: 24, SEQ ID NO. 25) of fibrolase (including the prepro region) being modified to create NAT. The N-terminal and C-terminal regions of mature fibrolase are indicated by underlining of terminal amino acid sequences (QQRF and LNKP) (nucleotides 262-265 and 461-464 of SEQ ID NO: 14). The N-terminal region (QQRF) is the one being modified (to substitute S for QQR). For oligos 3 and 4, below, dashed lines are inserted to denote the location of the omitted codons encoding for residues QQ in the N-terminal region of fibrolase.

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT ---------+---------+---------+---------+---------+---------+ TACTCTAAAGGAAGTTAAAAATGACGACAAAATAAGCGTCGTAGGAGGCGTAATCGACGA M  R  F  P  S  I  F  T  A  V  L  F  A  A  S  S  A  L  A  A CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT ---------+---------+---------+---------+---------+---------+ GGTCAGTTGTGATGTTGTCTTCTACTTTGCCGTGTTTAAGGCCGACTTCGACAGTAGCCA P  V  N  T  T  T  E  D  E  T  A  Q  I  P  A  E  A  V  I  G TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT ---------+---------+---------+---------+---------+---------+ ATGAGTCTAAATCTTCCCCTAAAGCTACAACGACAAAACGGTAAAAGGTTGTCGTGTTTA Y  S  D  L  E  G  D  F  D  V  A  V  L  P  F  S  N  S  T  N             Oligo 1     5′-TACTATTGCCAGCATTGCTGC-3′ AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA ---------+---------+---------+---------+---------+---------+ TTGCCCAATAACAAATATTTATGATGATAACGGTCGTAACGACGATTTCTTCTTCCCCAT N  G  L  L  F  I  N  T  T  I  A  S  I  A  A  K  E  E  G  V XhoI    | TCTCTCGAGAAAAGAGAGGCTGAAGCTTCTTCTATTATCTTGGAATCTGGTAACGTTAAC ---------+---------+---------+---------+---------+---------+ AGAGAGCTCTTTTCTCTCCGACTTCGAAGAAGATAATAGAACCTTAGACCATTGCAATTG S  L  E  K  R  E  A  E  A  S  S  I  I  L  E  S  G  N  V  N GATTACGAAGTTGTTTATCCAAGAAAGGTCACTCCAGTTCCTAGGGGTGCTGTTCAACCA ---------+---------+---------+---------+---------+---------+ CTAATGCTTCAACAAATAGGTTCTTTCCAGTGAGGTCAAGGATCCCCACGACAAGTTGGT D  Y  E  V  V  Y  P  R  K  V  T  P  V  P  R  G  A  V  Q  P AAGTACGAAGATGCCATGCAATACGAATTCAAGGTTAACAGTGAACCAGTTGTCTTGCAC ---------+---------+---------+---------+---------+---------+ TTCATGCTTCTACGGTACGTTATGCTTAAGTTCCAATTGTCACTTGGTCAACAGAACGTG K  Y  E  D  A  M  Q  Y  E  F  K  V  N  S  E  P  V  V  L  H TTGGAAAAAAACAAAGGTTTGTTCTCTGAAGATTACTCTGAAACTCATTACTCCCCAGAT ---------+---------+---------+---------+---------+---------+ AACCTTTTTTTGTTTCCAAACAAGAGACTTCTAATGAGACTTTGAGTAATGAGGGGTCTA L  E  K  N  K  G  L  F  S  E  D  Y  S  E  T  H  Y  S  P  D GGTAGAGAAATTACTACTTACCCATTGGGTGAAGATCACTGTTACTACCATGGTAGAATC ---------+---------+---------+---------+---------+---------+ CCATCTCTTTAATGATGAATGGGTAACCCACTTCTAGTGACAATGATGGTACCATCTTAG G  R  E  I  T  T  Y  P  L  G  E  D  H  C  Y  Y  H  G  R  I GAAAACGATGCTGACTCCACTGCTTCTATCTCTGCTTGTAACGGTTTGAAGGGTCATTTC ---------+---------+---------+---------+---------+---------+ CTTTTGCTACGACTGAGGTGACGAAGATAGAGACGAACATTGCCAAACTTCCCAGTAAAG E  N  D  A  D  S  T  A  S  I  S  A  C  N  G  L  K  G  H  F AAGTTGCAAGGTGAAATGTACTTGATTGAACCATTGGAATTGTCCGACTCTGAAGCCCAT ---------+---------+---------+---------+---------+---------+ TTCAACGTTCCACTTTACATGAACTAACTTGGTAACCTTAACAGGCTGAGACTTCGGGTA K  L  Q  G  E  M  Y  L  I  E  P  L  E  L  S  D  S  E  A  H GCTGTCTACAAGTACGAAAACGTCGAAAAGGAAGATGAAGCCCCAAAGATGTGTGGTGTT ---------+---------+---------+---------+---------+---------+ CGACAGATGTTCATGCTTTTGCAGCTTTTCCTTCTACTTCGGGGTTTCTACACACCACAA A  V  Y  K  Y  E  N  V  E  K  E  D  E  A  P  K  N  C  G  V                               Oligo 3  5′-TCCAATTAAACTTGACTAAG ACCCAAAACTGGGAATCATATGAACCAATCAAGAAGGCCTTCCAATTAAACTTGACTAAG ---------+---------+---------+---------+---------+---------+ TGGGTTTTGACCCTTAGTATACTTGGTTAGTTCTTCCGGAAGGTTAATTTGAACTGATTC                               Oligo 4   3′-GGTTAATTTGAACTGATTC T  Q  N  W  E  S  Y  E  P  I  K  K  A  F  Q  L  N  L  T  K AGA------TCTTTCCCACAAAGATACGTAC-3′ AGACAACAAAGATTCCCACAAAGATACGTACAGCTGGTTATCGTTGCTGACCACCGTATG ---------+---------+---------+---------+---------+---------+ TCTGTTGTTTCTAAGGGTGTTTCTATGCATGTCGACCAATAGCAACGACTGGTGGCATAC TCT------AGAAAGGGTGTTTCTATGCATG-5′ R  Q  Q  R  F  P  Q  R  Y  V  Q  L  V  I  V  A  D  H  R  M AACACTAAATACAACGGTGACTCTGACAAAATCCGTCAATGGGTGCACCAAATCGTCAAC ---------+---------+---------+---------+---------+---------+ TTGTGATTTATGTTGCCACTGAGACTGTTTTAGGCAGTTACCCACGTGGTTTAGCAGTTG N  T  K  Y  N  G  D  S  D  K  I  R  Q  W  V  H  Q  I  V  N ACCATTAACGAAATCTACAGACCACTGAACATCCAATTCACTTTGGTTGGTTTGGAAATC ---------+---------+---------+---------+---------+---------+ TGGTAATTGCTTTAGATGTCTGGTGACTTGTAGGTTAAGTGAAACCAACCAAACCTTTAG T  I  N  E  I  Y  R  P  L  N  I  Q  F  T  L  V  C  L  E  I TGGTCCAACCAAGATTTGATCACCGTTACTTCTGTATCCCACGACACTCTGGCATCCTTC ---------+---------+---------+---------+---------+---------+ ACCAGGTTGGTTCTAAACTAGTGGCAATGAAGACATAGGGTGCTGTGAGACCGTAGGAAG W  S  N  Q  D  L  I  T  V  T  S  V  S  H  D  T  L  A  S  F GGTAACTGGCGTGAAACCGACCTGCTGCGTCGCCAACGTCATGATAACGCTCAACTGCTG ---------+---------+---------+---------+---------+---------+ CCATTGACCGCACTTTGGCTGGACGACGCAGCGGTTGCAGTACTATTGCGAGTTGACGAC G  N  W  R  E  T  D  L  L  R  R  Q  R  H  D  N  A  Q  L  L ACCGCTATCGACTTCGACGGTGATACTGTTGGTCTGGCTTACGTTGGTGGCATGTGTCAA ---------+---------+---------+---------+---------+---------+ TGGCGATAGCTGAAGCTGCCACTATGACAACCAGACCGAATGCAACCACCGTACACAGTT T  A  I  D  F  D  G  D  T  V  G  L  A  Y  V  G  G  M  C  Q CTGAAACATTCTACTGGTGTTATCCAGGACCACTCCGCTATTAACCTGCTGGTTGCTCTG ---------+---------+---------+---------+---------+---------+ GACTTTGTAAGATGACCACAATAGGTCCTGGTGAGGCGATAATTGGACGACCAACGAGAC L  K  H  S  T  G  V  I  Q  D  H  S  A  I  N  L  L  V  A  L ACCATGGCACACGAACTGGGTCATAACCTGGGTATGAACCACGATGGCAACCAGTGTCAC ---------+---------+---------+---------+---------+---------+ TGGTACCGTGTGCTTGACCCAGTATTGGACCCATACTTGGTGCTACCGTTGGTCACAGTG T  M  A  H  E  L  G  H  N  L  G  M  N  H  D  G  N  Q  C  H TGCGGTGCAAACTCCTGTGTTATGGCTGCTATGCTGTCCGATCAACCATCCAAACTGTTC ---------+---------+---------+---------+---------+---------+ ACGCCACGTTTGAGGACACAATACCGACGATACGACAGGCTAGTTGGTAGGTTTGACAAG C  G  A  N  S  C  V  M  A  A  M  L  S  D  Q  P  S  K  L  F TCCGACTGCTCTAAGAAAGACTACCAGACCTTCCTGACCGTTAACAACCCGCAGTGTATC ---------+---------+---------+---------+---------+---------+ AGGCTGACGAGATTCTTTCTGATGGTCTGGAAGGACTGGCAATTGTTGGGCGTCACATAG S  D  C  S  K  K  D  Y  Q  T  F  L  T  V  N  N  P  Q  C  I               NotI                  | CTGAACAAACCGTAAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAAGAGGAT ---------+---------+---------+---------+---------+---------+ GACTTGTTTGGCATTCGCCGGCGGTCGAAAGATCTTGTTTTTGAGTAGAGTCTTCTCCTA L  N  K  P  *  A  A  A  S  F  L  E  Q  K  L  I  S  E  E  D CTGAATAGCGCCGTCCACGATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGAC ---------+---------+---------+---------+---------+---------+ GACTTATCGCGGCAGCTGGTAGTAGTAGTAGTAGTAACTCAAACATCGGAATCTGTACTG L  N  S  A  V  D  H  H  H  H  H  H  *  V  C  S  L  R  H  D TGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAG ---------+---------+---------+---------+---------+---------+ ACAAGGAGTCAAGTTCAACCCGTGAATGCTCTTCTGGCCAGAACGATCTAAGATTAGTTC C  S  S  V  Q  V  G  H  L  R  E  D  R  S  C  *  I  L  I  K AGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATT ---------+---------+---------+---------+---------+---------+ TCCTACAGTCTTACGGTAAACGGACTCTCTACGTCCGAAGTAAAAACTATGAAAAAATAA  CCTACAGTCTTACGGTAAACG-5′ Oligo 2 R  M  S  E  C  H  L  P  E  R  C  R  L  H  F  *  Y  F  F  I  -

EXAMPLE 2 Expression of NAT in Pichia pastoris

When attempts were made to express the DNA for NAT in E. coli, very poor refolding and a requirement for dilute conditions reduced the purification efficiency. These and other considerations led to the usage of Pichia pastoris, a yeast species, as the host cell. A culture of selected clones of Pichia pastoris which had been transfected with prepro NAT cDNA (SEQ ID NO: 4) was inoculated into 500 ml of the following inoculation growth medium:

Per liter of batch medium Yeast extract 30.0 g Potassium phosphate dibasic 17.2 g Glucose 20.0 g Biotin 0.004 g Water to 1 liter Phosphoric acid, 85% to adjust pH to 6.00

The transfected P. pastoris cells were incubated at 30° C. in a shaker for about 30 to 32 hours. About 1% (w/v) of the resulting culture was used to inoculate a 10-liter fermentor. The fermentor contained sterilized basal salts and glucose (below). Twelve milliliters per liter of PTM4 salts (PTM4 is a trace metals solution containing cupric sulfate pentahydrate, sodium iodide, manganese sulfate monohydrate, sodium molybdate dihydrate, boric acid, cobaltous chloride hexahydrate, zinc chloride, ferrous sulfate heptahydrate, d-biotin, sulfuric acid and purified water) were added per liter of batch medium after fermentor sterilization. The fermentation growth temperature was 30° C. The fermentor pH was controlled with ammonium hydroxide and phosphoric acid at pH 6.00. Zinc from the zinc salts added to the medium becomes incorporated into NAT as part of the metalloproteinase structure.

Basal salts per liter of batch medium Phosphoric acid, 85% 26.7 ml Calcium sulfate 0.93 g Potassium sulfate 18.2 g Magnesium sulfate-7H₂O 14.9 g Potassium hydroxide 4.13 g Glucose 30.0 g Water to 1 liter

The batch culture was grown until the glucose was completely consumed (17 to 20 hours). Then a fed-batch phase was initiated. The fed-batch phase media consisted of glucose and 12 ml of PTM4 salts per liter. The induction feed consisted of glucose, 25% methanol, and 12 ml of PTM4 salts per liter. At induction, the temperature of the reactor was shifted to 20° C. The induction phase lasted 60 to 75 hours. The conditioned media were harvested and the cellular debris was discarded.

EXAMPLE 3 Purification of NAT from Pichia pastoris

The yeast broth (conditioned media less cellular debris) from Example 2 was clarified and the pH and conductivity were adjusted to 6.5 and 10-20 mS/cm, respectively. The broth was loaded onto an immobilized metal affinity resin that had been charged with copper (Cu) and equilibrated with phosphate buffered saline (PBS). The resin was washed with PBS and eluted with an imidazole gradient (0-100 mN) in PBS. Fractions containing “mature” NAT (SEQ ID NO: 1) were pooled and diluted until the conductivity was less than 1.5 mS/cm, pH 6.4. The diluted pool was loaded onto an SP SEPHAROSE® high molecular weight resin for separation by gel filtration (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) that had been equilibrated with 10 mM 2-(N-morpholino) ethanesulfonic acid (MES). The column was washed with MES and eluted with a NaCl gradient (0-500 mM) in MES. Fractions containing NAT were pooled and stored.

EXAMPLE 4 Thrombolysis in Acute Thrombosis of Rat Carotid Artery; Comparison of NAT with Urokinase

To demonstrate that “mature” NAT (SEQ ID NO: 1) is biologically active and functionally unique, acute pharmacology studies were conducted in rats where focal injury to one of the carotid arteries was created by applying anodal current. This injury produces an occlusive thrombus which generally forms within fifteen minutes. Once the thrombus was formed, the artery was observed for a period of thirty minutes to assure that the carotid occlusion was stable. Then heparin and aspirin were administered intravenously to prevent further propagation of the thrombus. The animals were then treated with an intraarterial infusion of test material. Blood flow through the carotid artery was monitored during the delivery of test material so that successful clot lysis could be detected and the time at which clot lysis occurred could be noted. The percentage of experiments where clot lysis occurred was noted and group means were calculated for only those experiments where clot lysis was successful. As a measure of the hemorrhagic potential of the test material, any blood that was shed from the surgical site was collected with gauze swabs. The swabs were placed in a detergent solution to solubilize red blood cells and release hemoglobin, which was then quantified spectrophotometrically. Shed hemoglobin was used to calculate a volume of blood loss. Test data are reported in the Table below.

TABLE 1 Incidence of Clot Lysis, Time to Clot Lysis and Surgical Blood Loss (Mean ± std. dev.) INCIDENCE TIME TO OF LYSIS LYSIS BLOOD LOSS (%) (min) (ml) SALINE  0% N/A 0.10 ± (n = 6)  (0 of 6) 0.23 Urokinase 33% 55.3 ± 1.06 ± 25 U/min  (5 of 15) 15.9 1.59 (n = 15) Urokinase 86% 33.5 ± 1.43 ± 250 U/min (13 of 15) 15.3 1.45 (n = 15) NAT 2 mg 78% 6.3 ± 0.96 ± (n = 14) (11 of 14) 5.8 0.77

These studies establish that NAT is biologically active in an animal model of in vivo clot lysis. Further, clot lysis was achieved in a markedly reduced amount of time and with less blood loss from the surgical site, in comparison with urokinase. Thus, the activity profile of NAT can be distinguished from the plasminogen activator class of thrombolytic agents (represented by urokinase) in that clot lysis with NAT occurs more rapidly and with reduced hemorrhagic complications.

The fibrinolytic activity of NAT is comparable to that of fibrolase. In addition, as mentioned above the stability of the N-terminus of NAT results in a more homogeneous end product upon recombinant expression, which is a distinct advantage (i.e., the N-terminus will not change over time resulting in a mixture of different forms, thus making the polypeptide more stable).

                   #             SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 29 <210> SEQ ID NO 1 <211> LENGTH: 201 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: NAT (analog of fibrolase  #of Agkistrodon       Contourtrix) <400> SEQUENCE: 1 Ser Phe Pro Gln Arg Tyr Val Gln Leu Val Il #e Val Ala Asp His Arg 1               5    #                10   #                15 Met Asn Thr Lys Tyr Asn Gly Asp Ser Asp Ly #s Ile Arg Gln Trp Val             20       #            25       #            30 His Gln Ile Val Asn Thr Ile Asn Glu Ile Ty #r Arg Pro Leu Asn Ile         35           #        40           #        45 Gln Phe Thr Leu Val Gly Leu Glu Ile Trp Se #r Asn Gln Asp Leu Ile     50               #    55               #    60 Thr Val Thr Ser Val Ser His Asp Thr Leu Al #a Ser Phe Gly Asn Trp 65                   #70                   #75                   #80 Arg Glu Thr Asp Leu Leu Arg Arg Gln Arg Hi #s Asp Asn Ala Gln Leu                 85   #                90   #                95 Leu Thr Ala Ile Asp Phe Asp Gly Asp Thr Va #l Gly Leu Ala Tyr Val             100       #           105       #           110 Gly Gly Met Cys Gln Leu Lys His Ser Thr Gl #y Val Ile Gln Asp His         115           #       120           #       125 Ser Ala Ile Asn Leu Leu Val Ala Leu Thr Me #t Ala His Glu Leu Gly     130               #   135               #   140 His Asn Leu Gly Met Asn His Asp Gly Asn Gl #n Cys His Cys Gly Ala 145                 1 #50                 1 #55                 1 #60 Asn Ser Cys Val Met Ala Ala Met Leu Ser As #p Gln Pro Ser Lys Leu                 165   #               170   #               175 Phe Ser Asp Cys Ser Lys Lys Asp Tyr Gln Th #r Phe Leu Thr Val Asn             180       #           185       #           190 Asn Pro Gln Cys Ile Leu Asn Lys Pro         195           #       200 <210> SEQ ID NO 2 <211> LENGTH: 603 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Native pro-NAT (analog of # fibrolase) <400> SEQUENCE: 2 tctttcccac aaagatacgt acagctggtt atcgttgctg accaccgtat ga #acactaaa     60 tacaacggtg actctgacaa aatccgtcaa tgggtgcacc aaatcgtcaa ca #ccattaac    120 gaaatctaca gaccactgaa catccaattc actttggttg gtttggaaat ct #ggtccaac    180 caagatttga tcaccgttac ttctgtatcc cacgacactc tggcatcctt cg #gtaactgg    240 cgtgaaaccg acctgctgcg tcgccaacgt catgataacg ctcaactgct ga #ccgctatc    300 gacttcgacg gtgatactgt tggtctggct tacgttggtg gcatgtgtca ac #tgaaacat    360 tctactggtg ttatccagga ccactccgct attaacctgc tggttgctct ga #ccatggca    420 cacgaactgg gtcataacct gggtatgaac cacgatggca accagtgtca ct #gcggtgca    480 aactcctgtg ttatggctgc tatgctgtcc gatcaaccat ccaaactgtt ct #ccgactgc    540 tctaagaaag actaccagac cttcctgacc gttaacaacc cgcagtgtat cc #tgaacaaa    600 ccg                   #                   #                   #            603 <210> SEQ ID NO 3 <211> LENGTH: 462 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Native pro-NAT (analog of # fibrolase) <400> SEQUENCE: 3 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le #u Phe Ala Ala Ser Ser 1               5    #                10   #                15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl #u Asp Glu Thr Ala Gln             20       #            25       #            30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser As #p Leu Glu Gly Asp Phe         35           #        40           #        45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th #r Asn Asn Gly Leu Leu     50               #    55               #    60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al #a Lys Glu Glu Gly Val 65                   #70                   #75                   #80 Ser Leu Glu Lys Arg Glu Ala Glu Ala Ser Se #r Ile Ile Leu Glu Ser                 85   #                90   #                95 Gly Asn Val Asn Asp Tyr Glu Val Val Tyr Pr #o Arg Lys Val Thr Pro             100       #           105       #           110 Val Pro Arg Gly Ala Val Gln Pro Lys Tyr Gl #u Asp Ala Met Gln Tyr         115           #       120           #       125 Glu Phe Lys Val Asn Ser Glu Pro Val Val Le #u His Leu Glu Lys Asn     130               #   135               #   140 Lys Gly Leu Phe Ser Glu Asp Tyr Ser Glu Th #r His Tyr Ser Pro Asp 145                 1 #50                 1 #55                 1 #60 Gly Arg Glu Ile Thr Thr Tyr Pro Leu Gly Gl #u Asp His Cys Tyr Tyr                 165   #               170   #               175 His Gly Arg Ile Glu Asn Asp Ala Asp Ser Th #r Ala Ser Ile Ser Ala             180       #           185       #           190 Cys Asn Gly Leu Lys Gly His Phe Lys Leu Gl #n Gly Glu Met Tyr Leu         195           #       200           #       205 Ile Glu Pro Leu Glu Leu Ser Asp Ser Glu Al #a His Ala Val Tyr Lys     210               #   215               #   220 Tyr Glu Asn Val Glu Lys Glu Asp Glu Ala Pr #o Lys Met Cys Gly Val 225                 2 #30                 2 #35                 2 #40 Thr Gln Asn Trp Glu Ser Tyr Glu Pro Ile Ly #s Lys Ala Phe Gln Leu                 245   #               250   #               255 Asn Leu Thr Lys Arg Ser Phe Pro Gln Arg Ty #r Val Gln Leu Val Ile             260       #           265       #           270 Val Ala Asp His Arg Met Asn Thr Lys Tyr As #n Gly Asp Ser Asp Lys         275           #       280           #       285 Ile Arg Gln Trp Val His Gln Ile Val Asn Th #r Ile Asn Glu Ile Tyr     290               #   295               #   300 Arg Pro Leu Asn Ile Gln Phe Thr Leu Val Gl #y Leu Glu Ile Trp Ser 305                 3 #10                 3 #15                 3 #20 Asn Gln Asp Leu Ile Thr Val Thr Ser Val Se #r His Asp Thr Leu Ala                 325   #               330   #               335 Ser Phe Gly Asn Trp Arg Glu Thr Asp Leu Le #u Arg Arg Gln Arg His             340       #           345       #           350 Asp Asn Ala Gln Leu Leu Thr Ala Ile Asp Ph #e Asp Gly Asp Thr Val         355           #       360           #       365 Gly Leu Ala Tyr Val Gly Gly Met Cys Gln Le #u Lys His Ser Thr Gly     370               #   375               #   380 Val Ile Gln Asp His Ser Ala Ile Asn Leu Le #u Val Ala Leu Thr Met 385                 3 #90                 3 #95                 4 #00 Ala His Glu Leu Gly His Asn Leu Gly Met As #n His Asp Gly Asn Gln                 405   #               410   #               415 Cys His Cys Gly Ala Asn Ser Cys Val Met Al #a Ala Met Leu Ser Asp             420       #           425       #           430 Gln Pro Ser Lys Leu Phe Ser Asp Cys Ser Ly #s Lys Asp Tyr Gln Thr         435           #       440           #       445 Phe Leu Thr Val Asn Asn Pro Gln Cys Ile Le #u Asn Lys Pro     450               #   455               #   460 <210> SEQ ID NO 4 <211> LENGTH: 1386 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Encodes pro-NAT (analog o #f fibrolase) <400> SEQUENCE: 4 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc at #tagctgct     60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tg #tcatcggt    120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa ca #gcacaaat    180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga ag #aaggggta    240 tctctcgaga aaagagaggc tgaagcttct tctattatct tggaatctgg ta #acgttaac    300 gattacgaag ttgtttatcc aagaaaggtc actccagttc ctaggggtgc tg #ttcaacca    360 aagtacgaag atgccatgca atacgaattc aaggttaaca gtgaaccagt tg #tcttgcac    420 ttggaaaaaa acaaaggttt gttctctgaa gattactctg aaactcatta ct #ccccagat    480 ggtagagaaa ttactactta cccattgggt gaagatcact gttactacca tg #gtagaatc    540 gaaaacgatg ctgactccac tgcttctatc tctgcttgta acggtttgaa gg #gtcatttc    600 aagttgcaag gtgaaatgta cttgattgaa ccattggaat tgtccgactc tg #aagcccat    660 gctgtctaca agtacgaaaa cgtcgaaaag gaagatgaag ccccaaagat gt #gtggtgtt    720 acccaaaact gggaatcata tgaaccaatc aagaaggcct tccaattaaa ct #tgactaag    780 agatctttcc cacaaagata cgtacagctg gttatcgttg ctgaccaccg ta #tgaacact    840 aaatacaacg gtgactctga caaaatccgt caatgggtgc accaaatcgt ca #acaccatt    900 aacgaaatct acagaccact gaacatccaa ttcactttgg ttggtttgga aa #tctggtcc    960 aaccaagatt tgatcaccgt tacttctgta tcccacgaca ctctggcatc ct #tcggtaac   1020 tggcgtgaaa ccgacctgct gcgtcgccaa cgtcatgata acgctcaact gc #tgaccgct   1080 atcgacttcg acggtgatac tgttggtctg gcttacgttg gtggcatgtg tc #aactgaaa   1140 cattctactg gtgttatcca ggaccactcc gctattaacc tgctggttgc tc #tgaccatg   1200 gcacacgaac tgggtcataa cctgggtatg aaccacgatg gcaaccagtg tc #actgcggt   1260 gcaaactcct gtgttatggc tgctatgctg tccgatcaac catccaaact gt #tctccgac   1320 tgctctaaga aagactacca gaccttcctg accgttaaca acccgcagtg ta #tcctgaac   1380 aaaccg                  #                   #                   #         1386 <210> SEQ ID NO 5 <211> LENGTH: 203 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native fibrolase of Agkis #trodon contortrix <400> SEQUENCE: 5 Gln Gln Arg Phe Pro Gln Arg Tyr Val Gln Le #u Val Ile Val Ala Asp 1               5    #                10   #                15 His Arg Met Asn Thr Lys Tyr Asn Gly Asp Se #r Asp Lys Ile Arg Gln             20       #            25       #            30 Trp Val His Gln Ile Val Asn Thr Ile Asn Gl #u Ile Tyr Arg Pro Leu         35           #        40           #        45 Asn Ile Gln Phe Thr Leu Val Gly Leu Glu Il #e Trp Ser Asn Gln Asp     50               #    55               #    60 Leu Ile Thr Val Thr Ser Val Ser His Asp Th #r Leu Ala Ser Phe Gly 65                   #70                   #75                   #80 Asn Trp Arg Glu Thr Asp Leu Leu Arg Arg Gl #n Arg His Asp Asn Ala                 85   #                90   #                95 Gln Leu Leu Thr Ala Ile Asp Phe Asp Gly As #p Thr Val Gly Leu Ala             100       #           105       #           110 Tyr Val Gly Gly Met Cys Gln Leu Lys His Se #r Thr Gly Val Ile Gln         115           #       120           #       125 Asp His Ser Ala Ile Asn Leu Leu Val Ala Le #u Thr Met Ala His Glu     130               #   135               #   140 Leu Gly His Asn Leu Gly Met Asn His Asp Gl #y Asn Gln Cys His Cys 145                 1 #50                 1 #55                 1 #60 Gly Ala Asn Ser Cys Val Met Ala Ala Met Le #u Ser Asp Gln Pro Ser                 165   #               170   #               175 Lys Leu Phe Ser Asp Cys Ser Lys Lys Asp Ty #r Gln Thr Phe Leu Thr             180       #           185       #           190 Val Asn Asn Pro Gln Cys Ile Leu Asn Lys Pr #o         195           #       200 <210> SEQ ID NO 6 <211> LENGTH: 609 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <400> SEQUENCE: 6 caacaaagat tcccacaaag atacgtacag ctggttatcg ttgctgacca cc #gtatgaac     60 actaaataca acggtgactc tgacaaaatc cgtcaatggg tgcaccaaat cg #tcaacacc    120 attaacgaaa tctacagacc actgaacatc caattcactt tggttggttt gg #aaatctgg    180 tccaaccaag atttgatcac cgttacttct gtatcccacg acactctggc at #ccttcggt    240 aactggcgtg aaaccgacct gctgcgtcgc caacgtcatg ataacgctca ac #tgctgacc    300 gctatcgact tcgacggtga tactgttggt ctggcttacg ttggtggcat gt #gtcaactg    360 aaacattcta ctggtgttat ccaggaccac tccgctatta acctgctggt tg #ctctgacc    420 atggcacacg aactgggtca taacctgggt atgaaccacg atggcaacca gt #gtcactgc    480 ggtgcaaact cctgtgttat ggctgctatg ctgtccgatc aaccatccaa ac #tgttctcc    540 gactgctcta agaaagacta ccagaccttc ctgaccgtta acaacccgca gt #gtatcctg    600 aacaaaccg                 #                   #                   #        609 <210> SEQ ID NO 7 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <400> SEQUENCE: 7 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc at #tagctgct     60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tg #tcatcggt    120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa ca #gcacaaat    180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga ag #aaggggta    240 tctctcgaga aaagagaggc tgaagcttct tctattatct tggaatctgg ta #acgttaac    300 gattacgaag ttgtttatcc aagaaaggtc actccagttc ctaggggtgc tg #ttcaacca    360 aagtacgaag atgccatgca atacgaattc aaggttaaca gtgaaccagt tg #tcttgcac    420 ttggaaaaaa acaaaggttt gttctctgaa gattactctg aaactcatta ct #ccccagat    480 ggtagagaaa ttactactta cccattgggt gaagatcact gttactacca tg #gtagaatc    540 gaaaacgatg ctgactccac tgcttctatc tctgcttgta acggtttgaa gg #gtcatttc    600 aagttgcaag gtgaaatgta cttgattgaa ccattggaat tgtccgactc tg #aagcccat    660 gctgtctaca agtacgaaaa cgtcgaaaag gaagatgaag ccccaaagat gt #gtggtgtt    720 acccaaaact gggaatcata tgaaccaatc aagaaggcct tccaattaaa ct #tgactaag    780 agacaacaaa gattcccaca aagatacgta cagctggtta tcgttgctga cc #accgtatg    840 aacactaaat acaacggtga ctctgacaaa atccgtcaat gggtgcacca aa #tcgtcaac    900 accattaacg aaatctacag accactgaac atccaattca ctttggttgg tt #tggaaatc    960 tggtccaacc aagatttgat caccgttact tctgtatccc acgacactct gg #catccttc   1020 ggtaactggc gtgaaaccga cctgctgcgt cgccaacgtc atgataacgc tc #aactgctg   1080 accgctatcg acttcgacgg tgatactgtt ggtctggctt acgttggtgg ca #tgtgtcaa   1140 ctgaaacatt ctactggtgt tatccaggac cactccgcta ttaacctgct gg #ttgctctg   1200 accatggcac acgaactggg tcataacctg ggtatgaacc acgatggcaa cc #agtgtcac   1260 tgcggtgcaa actcctgtgt tatggctgct atgctgtccg atcaaccatc ca #aactgttc   1320 tccgactgct ctaagaaaga ctaccagacc ttcctgaccg ttaacaaccc gc #agtgtatc   1380 ctgaacaaac cg               #                   #                   #     1392 <210> SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide <400> SEQUENCE: 8 tactattgcc agcattgctg c            #                   #                   #21 <210> SEQ ID NO 9 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide <400> SEQUENCE: 9 gcaaatggca ttctgacatc c            #                   #                   #21 <210> SEQ ID NO 10 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide <400> SEQUENCE: 10 tccaattaaa cttgactaag agatctttcc cacaaagata cgtac    #                   #45 <210> SEQ ID NO 11 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide <400> SEQUENCE: 11 gtacgtatct ttgtgggaaa gatctcttag tcaagtttaa ttgg    #                   # 44 <210> SEQ ID NO 12 <211> LENGTH: 1620 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1620) <223> OTHER INFORMATION: Complementary (sense) strand # of antisense       strand (See SEQ ID NO:13 <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Coding sequence of native # pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 12 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc at #tagctgct     60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tg #tcatcggt    120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa ca #gcacaaat    180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga ag #aaggggta    240 tctctcgaga aaagagaggc tgaagcttct tctattatct tggaatctgg ta #acgttaac    300 gattacgaag ttgtttatcc aagaaaggtc actccagttc ctaggggtgc tg #ttcaacca    360 aagtacgaag atgccatgca atacgaattc aaggttaaca gtgaaccagt tg #tcttgcac    420 ttggaaaaaa acaaaggttt gttctctgaa gattactctg aaactcatta ct #ccccagat    480 ggtagagaaa ttactactta cccattgggt gaagatcact gttactacca tg #gtagaatc    540 gaaaacgatg ctgactccac tgcttctatc tctgcttgta acggtttgaa gg #gtcatttc    600 aagttgcaag gtgaaatgta cttgattgaa ccattggaat tgtccgactc tg #aagcccat    660 gctgtctaca agtacgaaaa cgtcgaaaag gaagatgaag ccccaaagat gt #gtggtgtt    720 acccaaaact gggaatcata tgaaccaatc aagaaggcct tccaattaaa ct #tgactaag    780 agacaacaaa gattcccaca aagatacgta cagctggtta tcgttgctga cc #accgtatg    840 aacactaaat acaacggtga ctctgacaaa atccgtcaat gggtgcacca aa #tcgtcaac    900 accattaacg aaatctacag accactgaac atccaattca ctttggttgg tt #tggaaatc    960 tggtccaacc aagatttgat caccgttact tctgtatccc acgacactct gg #catccttc   1020 ggtaactggc gtgaaaccga cctgctgcgt cgccaacgtc atgataacgc tc #aactgctg   1080 accgctatcg acttcgacgg tgatactgtt ggtctggctt acgttggtgg ca #tgtgtcaa   1140 ctgaaacatt ctactggtgt tatccaggac cactccgcta ttaacctgct gg #ttgctctg   1200 accatggcac acgaactggg tcataacctg ggtatgaacc acgatggcaa cc #agtgtcac   1260 tgcggtgcaa actcctgtgt tatggctgct atgctgtccg atcaaccatc ca #aactgttc   1320 tccgactgct ctaagaaaga ctaccagacc ttcctgaccg ttaacaaccc gc #agtgtatc   1380 ctgaacaaac cgtaagcggc cgccagcttt ctagaacaaa aactcatctc ag #aagaggat   1440 ctgaatagcg ccgtcgacca tcatcatcat catcattgag tttgtagcct ta #gacatgac   1500 tgttcctcag ttcaagttgg gcacttacga gaagaccggt cttgctagat tc #taatcaag   1560 aggatgtcag aatgccattt gcctgagaga tgcaggcttc atttttgata ct #tttttatt   1620 <210> SEQ ID NO 13 <211> LENGTH: 1620 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1620) <223> OTHER INFORMATION: Complementary (antisense) st #rand of sense       strand (See SEQ ID NO:12 <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Anti-coding sequence of n #ative pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 13 aataaaaaag tatcaaaaat gaagcctgca tctctcaggc aaatggcatt ct #gacatcct     60 cttgattaga atctagcaag accggtcttc tcgtaagtgc ccaacttgaa ct #gaggaaca    120 gtcatgtcta aggctacaaa ctcaatgatg atgatgatga tggtcgacgg cg #ctattcag    180 atcctcttct gagatgagtt tttgttctag aaagctggcg gccgcttacg gt #ttgttcag    240 gatacactgc gggttgttaa cggtcaggaa ggtctggtag tctttcttag ag #cagtcgga    300 gaacagtttg gatggttgat cggacagcat agcagccata acacaggagt tt #gcaccgca    360 gtgacactgg ttgccatcgt ggttcatacc caggttatga cccagttcgt gt #gccatggt    420 cagagcaacc agcaggttaa tagcggagtg gtcctggata acaccagtag aa #tgtttcag    480 ttgacacatg ccaccaacgt aagccagacc aacagtatca ccgtcgaagt cg #atagcggt    540 cagcagttga gcgttatcat gacgttggcg acgcagcagg tcggtttcac gc #cagttacc    600 gaaggatgcc agagtgtcgt gggatacaga agtaacggtg atcaaatctt gg #ttggacca    660 gatttccaaa ccaaccaaag tgaattggat gttcagtggt ctgtagattt cg #ttaatggt    720 gttgacgatt tggtgcaccc attgacggat tttgtcagag tcaccgttgt at #ttagtgtt    780 catacggtgg tcagcaacga taaccagctg tacgtatctt tgtgggaatc tt #tgttgtct    840 cttagtcaag tttaattgga aggccttctt gattggttca tatgattccc ag #ttttgggt    900 aacaccacac atctttgggg cttcatcttc cttttcgacg ttttcgtact tg #tagacagc    960 atgggcttca gagtcggaca attccaatgg ttcaatcaag tacatttcac ct #tgcaactt   1020 gaaatgaccc ttcaaaccgt tacaagcaga gatagaagca gtggagtcag ca #tcgttttc   1080 gattctacca tggtagtaac agtgatcttc acccaatggg taagtagtaa tt #tctctacc   1140 atctggggag taatgagttt cagagtaatc ttcagagaac aaacctttgt tt #ttttccaa   1200 gtgcaagaca actggttcac tgttaacctt gaattcgtat tgcatggcat ct #tcgtactt   1260 tggttgaaca gcacccctag gaactggagt gacctttctt ggataaacaa ct #tcgtaatc   1320 gttaacgtta ccagattcca agataataga agaagcttca gcctctcttt tc #tcgagaga   1380 taccccttct tctttagcag caatgctggc aatagtagta tttataaaca at #aacccgtt   1440 atttgtgctg ttggaaaatg gcaaaacagc aacatcgaaa tccccttcta aa #tctgagta   1500 accgatgaca gcttcagccg gaatttgtgc cgtttcatct tctgttgtag tg #ttgactgg   1560 agcagctaat gcggaggatg ctgcgaataa aacagcagta aaaattgaag ga #aatctcat   1620 <210> SEQ ID NO 14 <211> LENGTH: 464 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native pro-fibrolase of A #gkistrodon contortrix <400> SEQUENCE: 14 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le #u Phe Ala Ala Ser Ser 1               5    #                10   #                15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl #u Asp Glu Thr Ala Gln             20       #            25       #            30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser As #p Leu Glu Gly Asp Phe         35           #        40           #        45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th #r Asn Asn Gly Leu Leu     50               #    55               #    60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al #a Lys Glu Glu Gly Val 65                   #70                   #75                   #80 Ser Leu Glu Lys Arg Glu Ala Glu Ala Ser Se #r Ile Ile Leu Glu Ser                 85   #                90   #                95 Gly Asn Val Asn Asp Tyr Glu Val Val Tyr Pr #o Arg Lys Val Thr Pro             100       #           105       #           110 Val Pro Arg Gly Ala Val Gln Pro Lys Tyr Gl #u Asp Ala Met Gln Tyr         115           #       120           #       125 Glu Phe Lys Val Asn Ser Glu Pro Val Val Le #u His Leu Glu Lys Asn     130               #   135               #   140 Lys Gly Leu Phe Ser Glu Asp Tyr Ser Glu Th #r His Tyr Ser Pro Asp 145                 1 #50                 1 #55                 1 #60 Gly Arg Glu Ile Thr Thr Tyr Pro Leu Gly Gl #u Asp His Cys Tyr Tyr                 165   #               170   #               175 His Gly Arg Ile Glu Asn Asp Ala Asp Ser Th #r Ala Ser Ile Ser Ala             180       #           185       #           190 Cys Asn Gly Leu Lys Gly His Phe Lys Leu Gl #n Gly Glu Met Tyr Leu         195           #       200           #       205 Ile Glu Pro Leu Glu Leu Ser Asp Ser Glu Al #a His Ala Val Tyr Lys     210               #   215               #   220 Tyr Glu Asn Val Glu Lys Glu Asp Glu Ala Pr #o Lys Met Cys Gly Val 225                 2 #30                 2 #35                 2 #40 Thr Gln Asn Trp Glu Ser Tyr Glu Pro Ile Ly #s Lys Ala Phe Gln Leu                 245   #               250   #               255 Asn Leu Thr Lys Arg Gln Gln Arg Phe Pro Gl #n Arg Tyr Val Gln Leu             260       #           265       #           270 Val Ile Val Ala Asp His Arg Met Asn Thr Ly #s Tyr Asn Gly Asp Ser         275           #       280           #       285 Asp Lys Ile Arg Gln Trp Val His Gln Ile Va #l Asn Thr Ile Asn Glu     290               #   295               #   300 Ile Tyr Arg Pro Leu Asn Ile Gln Phe Thr Le #u Val Gly Leu Glu Ile 305                 3 #10                 3 #15                 3 #20 Trp Ser Asn Gln Asp Leu Ile Thr Val Thr Se #r Val Ser His Asp Thr                 325   #               330   #               335 Leu Ala Ser Phe Gly Asn Trp Arg Glu Thr As #p Leu Leu Arg Arg Gln             340       #           345       #           350 Arg His Asp Asn Ala Gln Leu Leu Thr Ala Il #e Asp Phe Asp Gly Asp         355           #       360           #       365 Thr Val Gly Leu Ala Tyr Val Gly Gly Met Cy #s Gln Leu Lys His Ser     370               #   375               #   380 Thr Gly Val Ile Gln Asp His Ser Ala Ile As #n Leu Leu Val Ala Leu 385                 3 #90                 3 #95                 4 #00 Thr Met Ala His Glu Leu Gly His Asn Leu Gl #y Met Asn His Asp Gly                 405   #               410   #               415 Asn Gln Cys His Cys Gly Ala Asn Ser Cys Va #l Met Ala Ala Met Leu             420       #           425       #           430 Ser Asp Gln Pro Ser Lys Leu Phe Ser Asp Cy #s Ser Lys Lys Asp Tyr         435           #       440           #       445 Gln Thr Phe Leu Thr Val Asn Asn Pro Gln Cy #s Ile Leu Asn Lys Pro     450               #   455               #   460 <210> SEQ ID NO 15 <211> LENGTH: 203 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native fibrolase of Agkis #trodon contortrix <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Xaa in position 1 repr #esents the chemical       compound, pyroglutamic acid.  This  #chemical compound is referred       to in the specification as the  #letter “E” <400> SEQUENCE: 15 Xaa Gln Arg Phe Pro Gln Arg Tyr Val Gln Le #u Val Ile Val Ala Asp 1               5    #                10   #                15 His Arg Met Asn Thr Lys Tyr Asn Gly Asp Se #r Asp Lys Ile Arg Gln             20       #            25       #            30 Trp Val His Gln Ile Val Asn Thr Ile Asn Gl #u Ile Tyr Arg Pro Leu         35           #        40           #        45 Asn Ile Gln Phe Thr Leu Val Gly Leu Glu Il #e Trp Ser Asn Gln Asp     50               #    55               #    60 Leu Ile Thr Val Thr Ser Val Ser His Asp Th #r Leu Ala Ser Phe Gly 65                   #70                   #75                   #80 Asn Trp Arg Glu Thr Asp Leu Leu Arg Arg Gl #n Arg His Asp Asn Ala                 85   #                90   #                95 Gln Leu Leu Thr Ala Ile Asp Phe Asp Gly As #p Thr Val Gly Leu Ala             100       #           105       #           110 Tyr Val Gly Gly Met Cys Gln Leu Lys His Se #r Thr Gly Val Ile Gln         115           #       120           #       125 Asp His Ser Ala Ile Asn Leu Leu Val Ala Le #u Thr Met Ala His Glu     130               #   135               #   140 Leu Gly His Asn Leu Gly Met Asn His Asp Gl #y Asn Gln Cys His Cys 145                 1 #50                 1 #55                 1 #60 Gly Ala Asn Ser Cys Val Met Ala Ala Met Le #u Ser Asp Gln Pro Ser                 165   #               170   #               175 Lys Leu Phe Ser Asp Cys Ser Lys Lys Asp Ty #r Gln Thr Phe Leu Thr             180       #           185       #           190 Val Asn Asn Pro Gln Cys Ile Leu Asn Lys Pr #o         195           #       200 <210> SEQ ID NO 16 <211> LENGTH: 202 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native fibrolase of Agkis #trodon contortrix <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Xaa in position 1 repr #esents the chemical       compound, pyroglutamic acid. This ch #emical compound is referred to       in the specification as the lette #r “E” <400> SEQUENCE: 16 Xaa Arg Phe Pro Gln Arg Tyr Val Gln Leu Va #l Ile Val Ala Asp His 1               5    #                10   #                15 Arg Met Asn Thr Lys Tyr Asn Gly Asp Ser As #p Lys Ile Arg Gln Trp             20       #            25       #            30 Val His Gln Ile Val Asn Thr Ile Asn Glu Il #e Tyr Arg Pro Leu Asn         35           #        40           #        45 Ile Gln Phe Thr Leu Val Gly Leu Glu Ile Tr #p Ser Asn Gln Asp Leu     50               #    55               #    60 Ile Thr Val Thr Ser Val Ser His Asp Thr Le #u Ala Ser Phe Gly Asn 65                   #70                   #75                   #80 Trp Arg Glu Thr Asp Leu Leu Arg Arg Gln Ar #g His Asp Asn Ala Gln                 85   #                90   #                95 Leu Leu Thr Ala Ile Asp Phe Asp Gly Asp Th #r Val Gly Leu Ala Tyr             100       #           105       #           110 Val Gly Gly Met Cys Gln Leu Lys His Ser Th #r Gly Val Ile Gln Asp         115           #       120           #       125 His Ser Ala Ile Asn Leu Leu Val Ala Leu Th #r Met Ala His Glu Leu     130               #   135               #   140 Gly His Asn Leu Gly Met Asn His Asp Gly As #n Gln Cys His Cys Gly 145                 1 #50                 1 #55                 1 #60 Ala Asn Ser Cys Val Met Ala Ala Met Leu Se #r Asp Gln Pro Ser Lys                 165   #               170   #               175 Leu Phe Ser Asp Cys Ser Lys Lys Asp Tyr Gl #n Thr Phe Leu Thr Val             180       #           185       #           190 Asn Asn Pro Gln Cys Ile Leu Asn Lys Pro         195           #       200 <210> SEQ ID NO 17 <211> LENGTH: 462 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Analog form of native  #pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 17 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le #u Phe Ala Ala Ser Ser 1               5    #                10   #                15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl #u Asp Glu Thr Ala Gln             20       #            25       #            30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser As #p Leu Glu Gly Asp Phe         35           #        40           #        45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th #r Asn Asn Gly Leu Leu     50               #    55               #    60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al #a Lys Glu Glu Gly Val 65                   #70                   #75                   #80 Ser Leu Glu Lys Arg Glu Ala Glu Ala Ser Se #r Ile Ile Leu Glu Ser                 85   #                90   #                95 Gly Asn Val Asn Asp Tyr Glu Val Val Tyr Pr #o Arg Lys Val Thr Pro             100       #           105       #           110 Val Pro Arg Gly Ala Val Gln Pro Lys Tyr Gl #u Asp Ala Met Gln Tyr         115           #       120           #       125 Glu Phe Lys Val Asn Ser Glu Pro Val Val Le #u His Leu Glu Lys Asn     130               #   135               #   140 Lys Gly Leu Phe Ser Glu Asp Tyr Ser Glu Th #r His Tyr Ser Pro Asp 145                 1 #50                 1 #55                 1 #60 Gly Arg Glu Ile Thr Thr Tyr Pro Leu Gly Gl #u Asp His Cys Tyr Tyr                 165   #               170   #               175 His Gly Arg Ile Glu Asn Asp Ala Asp Ser Th #r Ala Ser Ile Ser Ala             180       #           185       #           190 Cys Asn Gly Leu Lys Gly His Phe Lys Leu Gl #n Gly Glu Met Tyr Leu         195           #       200           #       205 Ile Glu Pro Leu Glu Leu Ser Asp Ser Glu Al #a His Ala Val Tyr Lys     210               #   215               #   220 Tyr Glu Asn Val Glu Lys Glu Asp Glu Ala Pr #o Lys Met Cys Gly Val 225                 2 #30                 2 #35                 2 #40 Thr Gln Asn Trp Glu Ser Tyr Glu Pro Ile Ly #s Lys Ala Phe Gln Leu                 245   #               250   #               255 Asn Leu Thr Lys Arg Ser Phe Pro Gln Arg Ty #r Val Gln Leu Val Ile             260       #           265       #           270 Val Ala Asp His Arg Met Asn Thr Lys Tyr As #n Gly Asp Ser Asp Lys         275           #       280           #       285 Ile Arg Gln Trp Val His Gln Ile Val Asn Th #r Ile Asn Glu Ile Tyr     290               #   295               #   300 Arg Pro Leu Asn Ile Gln Phe Thr Leu Val Gl #y Leu Glu Ile Trp Ser 305                 3 #10                 3 #15                 3 #20 Asn Gln Asp Leu Ile Thr Val Thr Ser Val Se #r His Asp Thr Leu Ala                 325   #               330   #               335 Ser Phe Gly Asn Trp Arg Glu Thr Asp Leu Le #u Arg Arg Gln Arg His             340       #           345       #           350 Asp Asn Ala Gln Leu Leu Thr Ala Ile Asp Ph #e Asp Gly Asp Thr Val         355           #       360           #       365 Gly Leu Ala Tyr Val Gly Gly Met Cys Gln Le #u Lys His Ser Thr Gly     370               #   375               #   380 Val Ile Gln Asp His Ser Ala Ile Asn Leu Le #u Val Ala Leu Thr Met 385                 3 #90                 3 #95                 4 #00 Ala His Glu Leu Gly His Asn Leu Gly Met As #n His Asp Gly Asn Gln                 405   #               410   #               415 Cys His Cys Gly Ala Asn Ser Cys Val Met Al #a Ala Met Leu Ser Asp             420       #           425       #           430 Gln Pro Ser Lys Leu Phe Ser Asp Cys Ser Ly #s Lys Asp Tyr Gln Thr         435           #       440           #       445 Phe Leu Thr Val Asn Asn Pro Gln Cys Ile Le #u Asn Lys Pro     450               #   455               #   460 <210> SEQ ID NO 18 <211> LENGTH: 602 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Fragment of fibrolase of  #Agkistrodon contortrix <400> SEQUENCE: 18 tactattgcc agcattgctg ctaaagaaga aggggtatct ctcgagaaaa ga #gaggctga     60 agcttcttct attatcttgg aatctggtaa cgttaacgat tacgaagttg tt #tatccaag    120 aaaggtcact ccagttccta ggggtgctgt tcaaccaaag tacgaagatg cc #atgcaata    180 cgaattcaag gttaacagtg aaccagttgt cttgcacttg gaaaaaaaca aa #ggtttgtt    240 ctctgaagat tactctgaaa ctcattactc cccagatggt agagaaatta ct #acttaccc    300 attgggtgaa gatcactgtt actaccatgg tagaatcgaa aacgatgctg ac #tccactgc    360 ttctatctct gcttgtaacg gtttgaaggg tcatttcaag ttgcaaggtg aa #atgtactt    420 gattgaacca ttggaattgt ccgactctga agcccatgct gtctacaagt ac #gaaaacgt    480 cgaaaaggaa gatgaagccc caaagatgtg tggtgttacc caaaactggg aa #tcatatga    540 accaatcaag aaggccttcc aattaaactt gactaagaga tctttcccac aa #agatacgt    600 ac                   #                   #                   #             602 <210> SEQ ID NO 19 <211> LENGTH: 816 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Fragment of fibrolase of  #Agkistrodon contortrix <400> SEQUENCE: 19 tccaattaaa cttgactaag agatctttcc cacaaagata cgtacagctg gt #tatcgttg     60 ctgaccaccg tatgaacact aaatacaacg gtgactctga caaaatccgt ca #atgggtgc    120 accaaatcgt caacaccatt aacgaaatct acagaccact gaacatccaa tt #cactttgg    180 ttggtttgga aatctggtcc aaccaagatt tgatcaccgt tacttctgta tc #ccacgaca    240 ctctggcatc cttcggtaac tggcgtgaaa ccgacctgct gcgtcgccaa cg #tcatgata    300 acgctcaact gctgaccgct atcgacttcg acggtgatac tgttggtctg gc #ttacgttg    360 gtggcatgtg tcaactgaaa cattctactg gtgttatcca ggaccactcc gc #tattaacc    420 tgctggttgc tctgaccatg gcacacgaac tgggtcataa cctgggtatg aa #ccacgatg    480 gcaaccagtg tcactgcggt gcaaactcct gtgttatggc tgctatgctg tc #cgatcaac    540 catccaaact gttctccgac tgctctaaga aagactacca gaccttcctg ac #cgttaaca    600 acccgcagtg tatcctgaac aaaccgtaag cggccgccag ctttctagaa ca #aaaactca    660 tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tg #agtttgta    720 gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac cg #gtcttgct    780 agattctaat caagaggatg tcagaatgcc atttgc       #                   #      816 <210> SEQ ID NO 20 <211> LENGTH: 1373 <212> TYPE: DNA <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Fragment of fibrolase of  #Agkistrodon contortrix <400> SEQUENCE: 20 tactattgcc agcattgctg ctaaagaaga aggggtatct ctcgagaaaa ga #gaggctga     60 agcttcttct attatcttgg aatctggtaa cgttaacgat tacgaagttg tt #tatccaag    120 aaaggtcact ccagttccta ggggtgctgt tcaaccaaag tacgaagatg cc #atgcaata    180 cgaattcaag gttaacagtg aaccagttgt cttgcacttg gaaaaaaaca aa #ggtttgtt    240 ctctgaagat tactctgaaa ctcattactc cccagatggt agagaaatta ct #acttaccc    300 attgggtgaa gatcactgtt actaccatgg tagaatcgaa aacgatgctg ac #tccactgc    360 ttctatctct gcttgtaacg gtttgaaggg tcatttcaag ttgcaaggtg aa #atgtactt    420 gattgaacca ttggaattgt ccgactctga agcccatgct gtctacaagt ac #gaaaacgt    480 cgaaaaggaa gatgaagccc caaagatgtg tggtgttacc caaaactggg aa #tcatatga    540 accaatcaag aaggccttcc aattaaactt gactaagaga tctttcccac aa #agatacgt    600 acagctggtt atcgttgctg accaccgtat gaacactaaa tacaacggtg ac #tctgacaa    660 aatccgtcaa tgggtgcacc aaatcgtcaa caccattaac gaaatctaca ga #ccactgaa    720 catccaattc actttggttg gtttggaaat ctggtccaac caagatttga tc #accgttac    780 ttctgtatcc cacgacactc tggcatcctt cggtaactgg cgtgaaaccg ac #ctgctgcg    840 tcgccaacgt catgataacg ctcaactgct gaccgctatc gacttcgacg gt #gatactgt    900 tggtctggct tacgttggtg gcatgtgtca actgaaacat tctactggtg tt #atccagga    960 ccactccgct attaacctgc tggttgctct gaccatggca cacgaactgg gt #cataacct   1020 gggtatgaac cacgatggca accagtgtca ctgcggtgca aactcctgtg tt #atggctgc   1080 tatgctgtcc gatcaaccat ccaaactgtt ctccgactgc tctaagaaag ac #taccagac   1140 cttcctgacc gttaacaacc cgcagtgtat cctgaacaaa ccgtaagcgg cc #gccagctt   1200 tctagaacaa aaactcatct cagaagagga tctgaatagc gccgtcgacc at #catcatca   1260 tcatcattga gtttgtagcc ttagacatga ctgttcctca gttcaagttg gg #cacttacg   1320 agaagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tg #c          1373 <210> SEQ ID NO 21 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Analog form of native  #pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 21 Lys Arg Arg Phe 1 <210> SEQ ID NO 22 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native pro-fibrolase of A #gkistrodon contortrix <400> SEQUENCE: 22 Ala Ala Ala Ser Phe Leu Glu Gln Lys Leu Il #e Ser Glu Glu Asp Leu 1               5    #                10   #                15 Asn Ser Ala Val Asp His His His His His Hi #s             20       #            25 <210> SEQ ID NO 23 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native pro-fibrolase of A #gkistrodon contortrix <400> SEQUENCE: 23 Val Cys Ser Leu Arg His Asp Cys Ser Ser Va #l Gln Val Gly His Leu 1               5    #                10   #                15 Arg Glu Asp Arg Ser Cys             20 <210> SEQ ID NO 24 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native pro-fibrolase of A #gkistrodon contortrix <400> SEQUENCE: 24 Ile Leu Ile Lys Arg Met Ser Glu Cys His Le #u Pro Glu Arg Cys Arg 1               5    #                10   #                15 Leu His Phe <210> SEQ ID NO 25 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Agkistrodon contortrix <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: Native pro-fibrolase of A #gkistrodon contortrix <400> SEQUENCE: 25 Tyr Phe Phe Ile 1 <210> SEQ ID NO 26 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Analog form of native  #pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 26 Ala Ala Ala Ser Phe Leu Glu Gln Lys Leu Il #e Ser Glu Glu Asp Leu 1               5    #                10   #                15 Asn Ser Ala Val Asp His His His His His Hi #s             20       #            25 <210> SEQ ID NO 27 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Analog form of native  #pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 27 Val Cys Ser Leu Arg His Asp Cys Ser Ser Va #l Gln Val Gly His Leu 1               5    #                10   #                15 Arg Glu Asp Arg Ser Cys             20 <210> SEQ ID NO 28 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Analog form of native  #pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 28 Ile Leu Ile Lys Arg Met Ser Glu Cys His Le #u Pro Glu Arg Cys Arg 1               5    #                10   #                15 Leu His Phe <210> SEQ ID NO 29 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Analog form of native  #pro-fibrolase of       Agkistrodon contortrix <400> SEQUENCE: 29 Tyr Phe Phe Ile 1 

What is claimed is:
 1. An isolated nucleic acid molecule encoding the polypeptide of SEQ ID NO:
 1. 2. The nucleic acid molecule of claim 1 which has the sequence of SEQ ID NO:
 4. 3. An expression vector comprising expression regulatory elements operatively linked to a nucleic acid molecule according to claim
 1. 4. An expression vector according to claim 3 in which the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:
 2. 5. A host cell transformed or transfected with the nucleic acid molecule of claim
 1. 6. A transformed or transfected host cell according to claim 5 which is a prokaryotic or eukaryotic cell.
 7. A transformed or transfected eukaryotic cell according to claim 6 which is a yeast cell.
 8. A transformed or transfected yeast cell according to claim 7 which is Pichia pastoris.
 9. A method for the production of NAT, a novel acting thrombolytic metalloproteinase encoded by the nucleic acid of claim 1, comprising culturing host cells containing a DNA molecule encoding said metalloproteinase operatively linked to regulatory elements for stimulating the expression thereof under conditions such that the DNA molecule is expressed, and isolating the expressed metalloproteinase from the host cell culture.
 10. The method of claim 9 in which the DNA molecule is of SEQ ID NO:
 4. 11. The method of claim 9 in which the host cells are yeast cells.
 12. The method of claim 11 in which the yeast cells are of Pichia pastoris.
 13. A method for the production of a metalloproteinase fibrinolytic agent, comprising culturing Pichia host cells containing a DNA molecule encoding NAT operatively linked to regulatory elements for stimulating the expression thereof under conditions such that the DNA molecule is expressed, and isolating the expressed metalloproteinase from the host cell culture.
 14. The method of claim 13 in which the Pichia host cell is Pichia pastoris. 